Draft PROGRESS REPORT: PRODUCT DEVELOPMENT OF CHIILED …€¦ · scrambled eggs, fish soup and...

Sea Urchin Project 1

Draft PROGRESS REPORT:

PRODUCT DEVELOPMENT OF CHIILED SEA URCHIN FOR HUMAN CONSUMPTION

Submitted to PHAMA

Dr Jimaima Lako

Consultant - Food Scientist

Date: March 2015


Executive Summary

The widely distributed edible sea urchin (cawaki) species that grows wild in Fiji is the Tripneustes gratilla species. This species is mainly harvested

near intertidal zones by women and are sold in the urban markets fresh either with shells or as the gonads packed in plastic containers. However

it has not been commercially processed for sale in supermarkets or for export. Sea urchin gonads are rich in proteins and a good source of

vitamin A and Bs, and are also believed to enhance virility. There has been no published research on the wild populations and catch data of sea

urchins in Fiji and hence further research is warranted to establish these. This project aimed at developing fresh-chilled sea urchin gonad products

and other high quality gonad products suitable for export.

Three separate processing trials were conducted during November 2014 to March 2015 on sea urchin species Tripneustes gratilla that were

harvested by Ministry of Fisheries officers from different locations in the Kalokolevu district to the west of Suva harbour. Harvesting was done

on the day of processing and the whole urchins stored in a commercial chiller prior to processing. Samples were processed, preserved using

selected preservatives, packed and stored in selected temperatures for shelf life determinations which included standard plate count, total

coliforms, psychrophiles, pH, salinity and physical characteristics such as colour, texture, flavour and taste. The preservatives used were dry salt,

salt solution or brine, alcohol and a solution containing dextrin, salt and sodium alginate. Storage temperature was varied between -5C to +5C

or ambient (approximately 25-30C) and of up to 62 days. Microbial, organoleptic and chemical analyses were conducted in determining the

shelf life of each product formulation and storage condition. The desirable organoleptic characteristics aimed at are bright mango-orange or

yellow colour, whole firm texture, fresh seaweed odour, fresh seaweed-sweet taste and free of leaking fluids.

Results showed that the most desirable and acceptable organoleptic characteristics were the gonads preserved in 5% dry salt stored at -5.40C

with the shelf life of 23 days and the 8% alcohol mixed with 5% dry salt stored at ambient temperature with the shelf life of 34 days. These

recommendations were obtained after evaluating the three trials of different processing procedures and formulation. It is interesting to note

that brining appeared to be unacceptable due to the oozing and leaking of yellow and orange fluid into the brine, contributing to unacceptable

milky-turbid solution. Soaking in sodium phosphate did not stop the oozing, instead aggravated milkiness and turbidity of brined samples hence,

the adoption of dry salting and alcohol based preservation formulations respectively.

Further work is required to test for consumer acceptance and for further inter-laboratory analyses to confirm the in-house and acceptability

tests. Further research is also needed to establish the ideal time and protocol for harvesting including temperature and storage conditions prior

to processing


1 . 0 I n t r o d u c t i o n

Sea urchins are marine benthic invertebrates that have calcareous shells and have moveable spines. These animals are generally found in the

mild to low intertidal zone; at depth up to about 50 meters (Reynolds and Wilen, 2000). There are many species of edible sea urchins, however

some common ones that are readily available and consumed in some parts of the world include red sea urchins; Strongylocentrotus franciscanus,

green sea urchins; Strongylocentrotus droebachien, purple sea urchins; Strongylocentrotus purpuratus, Evechinus chloroticus (endemic in New

Zealand), Psammechinus miliaris, Paracentrotus lividus (purple sea urchin) and Echinus esculentus (West-coast of Scotland) (Suckling et al., 2011).

In Fiji the widely distributed edible sea urchin is the Tripneustes gratilla species which grow wild and are harvested by women. Some of these

sea urchins are sold in the markets either in shells or as the gonads packed in plastic containers. Even though sea urchins are naturally known

to be important in reducing algal biomass by grazing on them and improving the health of the marine ecosystem, limited information is available

on its wild population and the catch data in Fiji.

In Japan and other European countries, sea urchin is regarded as one of the most valuable fishery products and highly prized commodity due its

unique flavour (Chen et al., 2013). Sea urchin gonad of both male and female are delicacies in many countries such as Japan, Korea, Greece,

France and New Zealand which are consumed in various ways such as eating raw in sushi or with lemon, onion, and olive oil, as flavours in omelettes,

scrambled eggs, fish soup and mayonnaise. Japan is the largest market of gonad in the world, as it is sold in sushi bars as delicacy (Sonu, 2003). In

Fiji and its neighbouring Pacific Island countries, sea urchin is not highly regarded compared to fin fish and sea cucumber, hence the development

of this product for the overseas market.

The price for sea urchin gonad is determined by its colour, quality, appearance and nutritional value and these factors are affected by season,

temperature, photoperiod and food intake as major factors (Chen et al., 2013; James et al., 2007; Schlosser et al., 2005). The colour of gonad is

an important criterion for marketability and obtaining high price in which bright mango orange or yellow colours being the most desirable. These

colours are derived primarily from various types of carotenoids present in them. Studies have shown that increasing the concentration levels of

carotenoids in the diets of sea urchin enhanced gonad quality and provided its preferred mango-orange colour (Shpigel et al., 2006). This means

that the diet of sea urchin (aquaculture) or what the sea urchins feed on (wild) determined the colour of gonads.

Furthermore, studies have identified the major carotenoids naturally found in sea urchin gonads. These include β-carotene, α-carotene, β-

echinenone, zeaxanthin, canthaxanxin, lutein, astaxanthin, diatoxanthin, fucoxanthin and alloxanthin (Shpigel et al., 2006). Of these, β-

echinenone had been discovered to develop and accumulate the bright yellow-orange or mango-orange coloured gonad. Apart from richness in


carotenoids, gonads of sea urchins are also shown to be a good source of energy, omega 3 fatty acids, polyunsaturated fatty acids, protein,

minerals (such as zinc) and vitamins (Suckling et al., 2011).

There are various ways of processing and preservation of sea urchins. These include fresh (chilled), salted, steamed, baked and frozen depending

on the market, distance and the mode of transportation (Kato and Schroeter, 1985).

This project aimed at developing fresh-chilled sea urchin products and other sea urchin gonad products suitable for export in collaboration with

Sai Yee Food Industries Ltd located in Wailada, Lami. Sai Yee Food Industries processes and export quite a number of fresh and vacuum packed

local root crops such as taro, cassava, breadfruit, yams, plantain; fruits and vegetables such as jackfruit, okra and duruka; and reef fish which it

purchases from villages and landowners that plant and grow them. The project was identified and prioritised by the Fiji Market Access Working

Group (MAWG) of the Australian Aid-funded Pacific Horticultural & Agricultural Market Access program (PHAMA).

2.0 Desirable Attributes for Sea Urchin Gonads

The most desirable attributes for sea urchin gonads that had been developed for export markets include; bright mango-orange or yellow colour,

whole firm texture, fresh seaweed odour, fresh seaweed-sweet taste, free of leaking fluids and high nutritional value (Shpigel et al., 2006). The

best size for individual pieces of gonad for packing ranges from 40-50mm in length (Kato, 1972; Kato and Schroeter, 1985).

Studies have revealed that orange gonads are obtained from male sea urchins while yellow gonads are from the female sea urchins. Dark brown

coloured gonads are thought to be degenerated gonads mainly due to starvation (Kato and Schroeter, 1985). Sea urchin gonad colour and flavour

quality correlated with the types of food consumed by sea urchins (Kato, 1972).

3.0 Summary of Processing and Preservation of Sea Urchin

Based on the gonads quality attributes discussed above the processing of sea urchins adopted the following flow chart procedures. Four

preservation methods were conducted as outlined in the flow chart below. Note the critical control points will be identified in the HACCP plan

that will proposed for each processing line for each product in the final report.


Receive raw fresh sea urchin. Store in the chiller (3-50C) Wash shell – with Cool (0-20C) sea water with 3.5% w/v) (S1) Shell split lengthwise to ensure gonad is not damaged and remove and discard digestive organs; sterilize tools and tables (S2)

Sort gonad by colour and remove left over intestinal lining with sterilized tools (S3) Take out gonads without damage and wash with 3.5% salt solution (3.5% w/v) - Don't touch by hands. Use sterilized tools (S4)

Wash gonads in 0-2.0oC salt solution (3.5% w/v). Change salt water regularly and maintain at 0-2.0oC Wash gonads repeatedly until no turbidity observed in salt water (3.5% at 0-2.0oC) (S5) Drain well - Keep its temperature at 0 -2.0oC with the use of ice

(S6)

Soak the gonads in the solution for 10sec

Dextrin & Alginate

Add 5% branched dextrin into 2.8% (w/v) sodium chloride and 0.3% sodium alginate

Add salt - 8% drained

gonads salt. Sprinkle salt on

the chopping board first.

Then lay out gonads on the

board and sprinkle

remaining salt on the

gonads.

Pack with salt

water in

containers

Add salt - 10% drained

gonads salt. Sprinkle salt on

the chopping board first.

Then lay out gonads on the

board and sprinkle

remaining salt on the

gonads.

Alcohol Brine Dry Salt


Seal and label

Store at -50C

Figure 1: Summary of Processing Flow Diagram of Sea Urchin

4.0 Gonad Yield

Gonad yield and its quality vary according to seasons. Studies on the lunar cycles showed that higher gonad yield is obtained during full moon

compared to last quarter moon (Manuel et al., 2013). At the last quarter moon, sea urchins are found to have been already spawned or were in

their spent conditions which contributed to low yields. The ideal test diameter of around 6-7 cm at 15 months are assumed to be sexually

matured in which spawning begins (Manuel et al., 2013; McManus et al., undated).

Further soak in 2.8% NaCl solution with 1.0% calcium chloride for another 10 sec

Mix and drain - Mix gonads and salt gently then keep for 30 -60 minutes.

Mix and drain - Mix gonads and salt gently then keep for 30 -60 minutes.

Drain well, pack in dry cool containers

Add 95% alcohol - Add

about 22% capacity of the

jar. Put in gonads gently. If

alcohol is not enough then

add some more alcohol.

Keep 30 minutes then put

the lid on the jar.

Pack in containers


In Fiji limited research related to the spawning of sea urchin is available. It is therefore important to note that unless the spawning time in Fiji is

fully established harvesting time would then be appropriately determined to ensure that high yield of gonads are obtained. However, the scope

of this research was beyond the study of sea urchin spawning. Nonetheless, based on the three different processing occasions conducted in Dec

17th 2014, Jan 14th 2015 and Feb 11th 2015, the highest yield was obtained in the January 14th processing. This may indicate that the spawning

time of sea urchin was around January 14th which was evident in the acceptable size of 8-10mm x 40-50mm and high quality gonads. Studies in

the Philippines revealed the peak spawning of T. gratilla species is from December to January in the Philippines (McManus et al., undated).

5.0 Quality Assurance

Processing of sea urchins for each preservation technique complied with the food safety regulation (Fiji Ministry of Health, 2010). This included

compliance of the good manufacturing practices (GMP) which included the cleaning protocol of the processing room and all contact surfaces

and utensils with 100ppm chlorine and sanitized with 75% alcohol before and after processing.

Processing of sea urchin was maintained below 50C of the 3.5% salt solution. Ice was continuously used at every station to retain the cold chain

below 50C as the operational limit. A temperature data logger was used in recording the temperature.

Shell split was done lengthwise to avoid and reduce damage to gonads due to oozing if the tissue was physically damaged. Sorting of gonad size

and colour was conducted after shell splitting to ensure reasonable gonad size with acceptable yellow and orange as shown in Figure 2 below

were retained while dark brown was rejected. Only full size of at least 8-10mm thickness (any length) gonads were accepted.


Figure 2: Yellow and Orange gonads with two types of shell splits a and b

6.0 Processing Trials, Preservations and Shelf Life Tests

The processed sea urchin gonads discussed in section 3.0 above were treated with various preservatives and stored at selected temperatures;

-50C, 00C, 50C and ambient temperature for a certain duration of time prior to microbial analyses, chemical and organoleptic tests in order to

determine shelf life or period of storage until the gonads are unfit for consumption.

A total of three processing trials of sea urchins were carried out on separate occasions as indicated below;

trial 1-Dec 17th 2014,

a. Lengthwise split showing yellow gonads b. Crosswise split showing orange gonads


trial 2 – Jan 14th 2015 and

trial 3 – Feb 11th 2015.

6.1 Trial 1 Processing

6.1.1. Processing Design and Protocol

Trial 1 processing involved the processing of 3 prototypes: preserved in 5.3% brine stored at 50C and 00C as well as in 21% alcohol stored at

ambient temperature as shown in the experimental flow diagram in Figure 3 below.

Figure 3: Trial 1 prototypes and storage temperatures

6.1.2 Shelf Life Prediction Test

The three prototypes processes in Figure 3 above were stored at the selected temperatures as indicated and tested for shelf life over a period

of 15 days using the following indicators; microbial determinations, organoleptic assessments and chemical analyses. These tests were carried

out on all the preserved samples stored at -0.40C, 0.70C and ambient temperature of 28.10C as indicated in Table 1 below. It is important to note

Trial 1: 3 prototypes

Packed in 21% alcohol Packed in 5.3% brine

Stored @ 00C Stored @ 50C Stored @ ambient temperature


these temperatures are only average values and that there had been high variations in the range of temperature recorded. The details of tests

with results are discussed below.

6.1.2.1 Microbial Determination

Three major microbial analyses; standard plate count, psychrophilic bacterial count and coliforms were employed in the shelf life prediction of

gonads preserved in 5.3% brine stored at 0.70C and -0.40C and the 21% alcohol stored at ambient temperature over a period of 15 days for the

trial 1 processing of gonads. E.coli was to be further analysed only when coliform levels were found high. Due to insignifant levels of coliform

(<3MPN/g) obtained in day 1, the test was discontinued at days 7 and 15 which indicated good quality control and safe processing conducted.

Table 1: Microbial Levels of Gonads from Processing Trial 1

Type of test 5.3% brine 21% alcohol stored @ ambient tempt (28.10C) Stored @ -0.40C Stored @ 0.70C

Day 1 Day 7 Day 15 Day 1 Day 7 Day 15 Day 1 Day 7 Day 15

SPC (Aerobic Plate Count) (CFU/g) or EAPC/g-<300)

1×102

(eapc)

2.1×102

(eapc) 5.1×102

2.1×102

(eapc) 1.8×102

(eapc) 7.7×102

2.2×103

2.0×103

2.9×102

(eapc)

Psychrophilic (CFU/g or EAPC/g-<2500)

2.6×104 1.5×104

2.1×103

(eapc) 9.7×103 9.8×103

3.0×103

6.6×103

1.5×102

(eapc) 1.0×102

(eapc)

Coliforms (MPN/g) <3 <3

<3

EPAC – estimated plate aerobic count is referred to <300 counts for Standard Plate Count (SPC) and <2500 counts for psychrophilic bacteria.

The microbial results shown in Table 1 revealed that even though the aerobic plate count increased over time, the levels were significantly lower

as evident in the count of <300, while the psychrophilic bacteria were reduced in count over the 15 day shelf life duration for the three protocols.

Results also showed that 5.3% brine stored at -0.40C had lower levels of microbes compared to 0.70C and much lower in 21% alcohol on day 15.

Furthermore, the day 1 microbial levels indicated the effectiveness in the compliance of the Good Manufacturing Practices (GMP) and the safe

processing methods in maintaining of <50C chilled of 3.5% brine solution which effectively controlled the growth of bacteria below 300 count of


SPC and retained the firmness of gonads as shown in 6.1.2.2 below. This may indicate that even though the microbial levels are low until day 15,

the organoleptic assessments discussed below appeared to be the major determining factor in the acceptance of the gonad products hence, the

importance of employing multiple indicators in the shelf life prediction protocol.

6.1.2.2 Organoleptic Assessments

The organoleptic assessments employed two techniques; the descriptive profiling and hedonic scaling of each protocol stored at various

temperatures indicated in Tables 2 and 3 below. Table 2 showed that preservation in 5.3% brine resulted in milky- turbid brine solution, however

-0.40C storage revealed the following descriptive profile of gonads; lighter turbidity fluid, bright firm gonads, retained strong seaweed odour and

flavour with some sweet taste up to day 7 compared to 0.70C storage with the following descriptive profile; intense milky –turbid fluid, formation

of film like on the surface of brine, soft gonads with weak seaweed odour and flavour and have lost the sweet taste. On day 15, the 0.70C stored

sample became dull in colour, softer texture with neutral odour and weak seaweed flavour compared to -0.40C sample had retained seaweed

flavour and sweet taste and just begun to get soft in texture and a bit taint in colour. Given these characteristics, the -0.40C storage is estimated

to have a shelf life of 10 days calculated as 70% of day 15 while 0.70C storage sample is estimated to have the shelf life of 5 days calculated as

70% of day 7. The 21% alcohol sample stored at room temperature had a shelf life of more than 15 days even though had strong alcohol flavour

and bitter aftertaste, the texture was more firmer, dark brighter in colour with sweet-alcohol odour.

Table 2: Descriptive Profile of Gonads from Processing Trial 1

Type of test

5.3% brine 21% alcohol @ stored ambient tempt (28.10C)

Stored @ -0.40C Stored @ 0.70C


Colour Milky-turbid

brine but

bright gonads

Milky-turbid

brine but

bright gonads

yellow-milky brine with a bit dull gonads

Intense milky-turbidity

Milky-turbid brine with film like on surface of brine

Milky brine with light brown dull gonads

Clear transparent liquid with bright color gonads

Light milky liquid with bright color

Less milky liquid with dark color


Texture Firm Firm Softer than day 7

Soft-melting

out

softer Soft Firm Firm Firm denatured gonads

Odor Strong fresh seaweed

Strong fresh seaweed

Fresh seaweed

Fresh seaweed

Weak seaweed

No odour Strong alcohol

Alcohol with sweet odour

Strong alcohol with sweet odour

Taste & Flavor

Strong seaweed flavor with sweet taste


Seaweed flavor with sweet taste


Weak seaweed salty flavor

Weak seaweed only

Strong alcohol

Alcohol Sweet-alcohol flavour

Aftertaste Sweet-seaweed

Sweet-seaweed

Sweet-seaweed

Sweet-seaweed

salty salty Bitter -alcohol

Bitter -alcohol

Bitter -alcohol

n=3-6

The hedonic scale of the three protocols shown in Table 3 revealed that on day 1, 5.3% brine stored at 0.70C was ranked 1st, 5.3% brine stored

at -0.40C ranked 2nd while 18% alcohol stored at 28.10C ranked 3rd. On day 7, 5.3% brine stored at -0.40C ranked 1st, while 18% stored at 28.10C

ranked 2nd and the 5.3% brine stored at 0.70C ranked 3rd. On day 15, the 18% alcohol stored at 28.10C ranked 1st, 5.3% brine stored at -0.40C

ranked 2nd while 5.3% brine stored at 0.70C ranked 3rd.

Table 3: Hedonic Scaling of Gonads from Processing Trial 1

Type of test 5.3% brine 21% alcohol @ stored ambient tempt (28.10C) Stored @ -0.40C Stored @ 0.70C


Colour 4 2 3.5 3 2 2.5 5 4 4

Odor 4 3 3.5 3.8 3 2 3.3 3 4

Texture 3.8 2 2.5 3 2 2.5 4.3 5 4.5

Taste 4.5 5 4 4.3 2 2 2 4 4

Flavour 4.8 5 4.5 4.8 2 2.5 2 3 3.5

Overall Ranking* 2nd 1st 2nd 1st 3rd 3rd 3rd 2nd 1st n=3-6, Overall ranking* was based on individual ranking from 1st -3rd of the 3 prototypes; 1st ranking, 2nd ranking and 3rd ranking


Based on the hedonic scale and the organoleptic assessments, the 0.70C storage condition and the use of brining were eliminated. However the

following recommendations were made for trial 2 processing protocols; the use of Sodium Phosphate (alum) to stop the oozing and bleeding of

gonads, both for the 5.3% brine and 10% dry salt and to be both stored at 00C and -50C respectively as well a reduction of alcohol concentration

to 14% and stored at 50C and ambient temperature respectively.

6.1.2.3 Chemical Tests

Further insights into the reactions taking place during storage, three chemical analyses were measured; water activity, salinity and pH as shown

in Table 4 below. Results showed that slight changes in pH, salinity and water activity that occurred. The 5.3% brine stored at -0.40C revealed a

slight reduction in water activity, a slight increase in pH and salinity over the 15 days of storage while the 5.3% brine stored at 0.70C slight

reduction in water activity, no change in salinity and an increase in pH. The 21% alcohol had much lower pH even though a slight increase in pH

was observed over the 15 day period.

Table 4: Chemical Analyses of Gonads from Processing Trial 1

Type of test 5.3% brine 18% alcohol @ ambient tempt (28.10C) Stored @ -0.40C Stored @ 0.70C


Water Activity 0.968

@24.50C

0.963 @250C

0.964 @24.80C

0.966 @23.90C

0.960 @25.10C

0.962 @24.30C

0.949 @25.60C

0.950 @25.80C

0.952 @25.60C

Salinity (%w/w) 7.7 7.7 7.9 8.3 7.7 8.3 >10.5 >10.5 >10.5

pH@250C 6.86 6.98 6.94 6.75 6.92 6.93 6.28 6.42 6.5

n=3

Based on the combined results of microbial, descriptive profile, hedonic scale and chemical analyses the following suggestions were made to

improve the quality of the tested products;

(a) The oozing and continuous bleeding of gonads that contributed to milkiness of the brine solution needed to be inhibited by the use of

Sodium Phosphate

(b) Confirmation temperatures for 5.3% brine stored at 00C and -50C respectively

(c) 10% dry salt to be developed for storage at 00C and -50C respectively.


(d) A reduction in alcohol concentration to 14% and then stored at two temperatures; 50C and ambient temperature respectively were to

be adopted.

The suggestions were carried as part of trial 2 processing as indicated below.


6.2.1 Trial 2 Processing Design

Trial 2 processing involved the comparison of 6 protoptypes: soaked in 0.7% Sodium Phosphate (alum) for 1 min and then preserved in 5.3%

brine and 10% dry salt prior to storage at 00C and -50C respectively; 14% alcohol then stored at 50C or ambient temperature respectively and

then all stored for up to a total of 62 days as shown in the experimental flow diagram in Figure 4 below.


Alum soaked & packed

in 5.3% brine

Alum soaked & packed

in 10% dry salt

Packed in 14% alcohol

Stored @ ambient

temperature

Stored @ 50C

Stored @ -50C

Stored @ 00C




The six protocols were tested for shelf life over a period of 62 days using similar indicators as in trial 1: microbial determinations (excluding

coliforms), organoleptic assessments and chemical analyses. These tests were carried out on preserved samples that were planned for storage

at -50C and 00C for both brined and dry salted samples while the 14% alcohol samples were stored at 50C and ambient temperature. However,

due to temperature problems related to such facilities at the University premises as confirmed by the temperature data logger, the temperatures

of the respective cool and cold storage facilities used were recorded as -1.50C, 1.70C and ambient temperature of 300C as indicated in Table 5

below. It is important to note these temperatures are only average values and that there had been high variations in the temperature range

obtained which suggests for better control in the next round of preservation.


Only two microbial analyses; standard plate count and psychrophilic bacterial count were conducted in the shelf life prediction of gonads

preserved in 5.3% brine and 10% dry salt stored at -1.50C and 1.70C respectively and as well as the 14% alcohol stored at 1.70C and at ambient

temperature respectively for a period of up to 62 days. Total coliform test was removed for trial 2 processing based on the insignifant levels

obtained in trial 1 processing. The presence of coliform in samples would signify poor hygienic practices and poor sanitation especially the

removal and cleaning of digestive and intestinal organs and waste protocol or may be due to cross contamination. Given the GMP compliance

and the strict observations of time and temperature control during processing, we were confident that coliform levels would be also insignificant.

Results as indicated in Table 5 below showed that microbial levels of gonads were generally low indicating <106 (Health Protection Agency, 2009)

from day 1 to day 43 of selected samples even though fluctuations in data was observed.



Type of test

Alum 5.3% brine Alum 10% dry salt 14% alcohol

@ -1.50C @ 1.70C @ -1.50C @ 1.70C @ 1.70C @ ambient tempt of 300C

D1 D15 D22 D1 D15 D22 D1 D15 D22 D29 D43 D62 D1 D15 D22 D29 D15 D22 D29 D43 D62 D1 D15 D22 D29 D43 D62

SPC (CFU/g) or EAPC/g-<300)

3.8 ×

102

3.4

×

102

1.3

×

102

4.0

×

102

2.5

×

102

2.6

×

102

2.7

×

103

2.6

×

102

1.3

×

103

2.0

×

102

5.5

×

102

2.3

×

105

6.7

×

102

2.6

×

102

6.2

×

102

1.9

×

102

5.3

×

103

8.7

×

102

1.1

×

104

4.3

×

102

4.8

×

103

2.3

×

103

4.4

×

103

1.9

×

103

2.7

×

102

3.3

×

103

6.4

×

103


3.5 ×

103

6.0

×

102

1.0

×

102

3.3

×

103

8.0

×

102

2.0

×

102

1.8

×

103

ND

3.0

×

102

1.5

×

102

50

1.0

×

106

1.5

×

102

NC

1.5

×

102

3.0

×

102

2.2

×

103

ND

ND

3.0

×

102

7.0

×

102

2.5

×

102

3.0

×

102

ND

ND

ND

50

ND = Detection limit is 100 colonies, EPAC – estimated plate aerobic count is referred to <300 counts for Standard Plate Count (SPC) and <2500 counts for

psychrophilic bacteria.

It is important to note that the 5.3% brine that was soaked in sodium phosphate for 1 min prior to storage at both -1.50C and 1.70C were both

eliminated after day 22 due to the unacceptable intense milky-turbid brine solution as indicated in Table 7. Similarly, samples soaked in sodium

phosphate preserved in 10% dry salt and stored at 1.70C was eliminated at day 29 due to unacceptable rotten banana odour. These may indicate

that even though the microbial levels were low until certain days such as day 43 and day 62 for some samples, the organoleptic assessments

discussed below appeared to be the major determining factor in the acceptance of the gonad products hence, the importance of employing

multiple indicators in the shelf life prediction protocol as mentioned earlier. Based on the observations carried out in trial 1 processing of the

21% alcohol stored at ambient temperature, trial 2 of day 1 of the 14% alcohol stored at 1.70C microbial analysis were not conducted. This was

due to the assumption that the combination effect of the preservatives used; 14% alcohol mixed with the 10% dry salt stored at 1.70C would be

sufficient to control the growth of microbes.



Similar to trial 1, organoleptic assessments employed included descriptive profiling and hedonic scaling of each prototype stored at various

temperatures indicated in Tables 7 and 8 below. Table 7 showed that soaking in 0.7% sodium phosphate (alum) appeared not to be effective as

it aggravated and intensified the milkiness and turbidity of the brine solution, and also contributed to bitter aftertaste and squizzed tongue. This

may mean that alum is not effective in controlling the oozing in the brine solution hence did not improve the appearance of the packaged

product. Even though texture and taste may have been improved to a certain degree, the appearance of the milky brine solution may not be

favourable to consumers which may be assumed to be a spoilt product, hence eliminated.

Table 7: Organoleptic Evaluation of Gonads from Descriptive Profile of Gonads from Processing Trial 2

Type of

test Alum 5.3% brine Alum 10% dry salt 14% alcohol

@ -1.50C @ 1.70C @ -1.50C @ 1.70C @ 1.70C @ ambient tempt (300C)


Colour Pale

orange

turbid

brine

Milky

turbid

brine

Light

milky

turbid

brine

Yello

w-

milk

y

brine

Very

milk

y

brine

with

fresh

brigh

t

gona

ds

Very

milky

turbi

d

brine

Brigh

t

yello

w

Light

color

Brigh

t

dark

brigh

t

Brigh

t

Milk

y

brigh

t

color

Fres

h

orna

ge

Light

color

Brigh

t

Bright Dar

k

colo

r

Dull Brigh

t

Brigh

t

Brigh

t

Brigh

t

yello

w

inten

se

Dark

color

Dull Dark Brigh

t

Brig

ht

Texture firm soft firm firm firm Meal

time

out

Meal

time

&

brea

k

easil

y

firm firm firm firm firm Firm

but

start

to

melt

firm Firm Getti

ng

soft

firm firm Firm Firm

coag

ulate

d

Firm Very

firm

coag

ulate

d

firm firm firm Firm

coag

ulate

d

Firm


Odor Fresh seaweed

neutral Mild seaweed

Strong fresh seaweed

seaweed

seaweed

neutral

seaweed

Neutral

Neutral

Sea weed

Neutral

neutral

neutral

Neutral

Rotten banana (ester)

alcohol

Neutral

Alcohol

Mild seaweed strong alcohol

Alcohol

Weak alcohol

alcohol

Mild alcohol

alcohol

Strong alcohol

Strong alcohol

Taste & Flavor

Fresh seaweed& salty

Very salty

Mild seaweed sweet salty

Fresh seaweed sweet & salty

Seaweed sweet salty

Seaweed sweet

Salty & sweet

Very salty

Salty Salty Seaweed sweet salty

Weak sea weed

Salty seaweed

salty Salty

Salty alcohol

Alcohol

Alcohol

Alcohol

Alcohol

Alcohol with sweet & salty

alcohol

alcohol

Alcohol

Strong alcohol and salty

Strong alcohol umami taste

Aftertaste

Squized tongue

astringency

Squized tongue

Prawn tail and bitter

Squizzed tongue

Squizzed tongue

bitter

Squizzed tongue

Squizzed tongue

Squizzed tongue

Sweet salty squizzed tongue

Slightly bitter

bitter

Squizzed tongue

Squizzed tongue

Squizzed tongue

alcohol

Alcohol

Alcohol

Alcohol

Alcohol

bitter

alcohol

Strong alcohol

Alcohol

Alcohol

Weak alcohol

Out of the six prototypes, only three reached the 62 days of shelf life testing. These were the 10% dry salt stored at -1.50C, the 14% alcohol

stored at both 1.70C and ambient temperature respectively that retained bright colour, firm texture and seaweed odour and flavour. However,

the 10% dry salt was too salty, the 14% alcohol stored at 1.70C had light milky liquid which may not be favourable to consumers and the 14%

alcohol stored at ambient temperature appeared to have the most favourable appearance even though with a bitter alcohol taste and strong

alcohol odour and flavour. These were confirmed by the hedonic scaling as indicated in Table 8 above which revealed that out of the six

prototypes, the 5.3% brine stored at 1.70C was ranked 1st both on days 1 and 15, the 10% dry salt stored at 1.70C was ranked 1st on days 22 and

29 while the 14% alcohol stored at ambient temperature ranked 1st on day 2.


Table 8: Hedonic Evaluation of Gonads from Processing Trial 2

Type of

test




Colour 3.7 3.5 2.5 4.2 4 1 3.2 3.5 4 3.7 4.5 3.0 4 3.5 4 4.3 3 3.5 3.8 4.5 4.0 4.5 3.5 3.5 4 4.5 4.5

Odor 4 3 4 4.3 3.5 3.5 2.8 2.5 3 3.5 4.5 3.5 3 2.5 3 4 2.5 3.5 3.0 3.0 3.5 3.8 3 3.5 3.2 3.0 4.0

Texture 3.5 2.5 3 4.3 3.5 2.5 3.3 2.5 3.5 3.3 4.5 3.5 3.7 3 3.5 4.7 4 4.5 4.3 4.5 4.0 4.5 4.5 4.5 3.8 4.5 4.5

Taste 3.8 3 3.5 4.2 3.5 3.5 3 2.5 2.5 2.3 4.5 3.5 2.2 2 2.5 4.2 2.5 2 2.7 3.0 3.0 4.2 3.5 1.5 3 2.5 4.0

Flavor 3.3 3 3 4.7 3.5 3 2.5 1.5 2.5 2.2 4.5 4.0 2.3 1.5 2.5 4 2.5 1.5 2.5 3.5 3.0 3.7 3 1.5 2.8 2.5 3.5

Overall ranking*

2nd 3rd 2nd 1st 1st 5th 4th 5th 3rd 4th 1st 3rd 5th 5th 1st 1st 4th 6th 2nd 2nd 2nd 3rd 2nd 4th 3rd 3rd 1st

n=3-6, Overall ranking* was based on individual ranking from 1st -3rd of the 6 prototypes; 1st ranking, 2nd ranking and 3rd ranking, etc

Based on the hedonic scale and the organoleptic assessments, the 5.3% brine soaked in sodium phosphate (alum) for 1 min prior to storage at

both -1.50C and 1.70C were both eliminated after day 22 which left the shelf life at 16 days. However due to unacceptable intense milky-turbid

brine solution and the effect of squizzed tongue eliminate the use of sodium phosphate. Similarly, soaked in alum 10% dry salt stored at 1.70C

was eliminated at day 29 due to unacceptable rotten banana odour which made the shelf life at 20 days, however due to tongue squizzed effect,

this sample may not be acceptable. This means that based on physical appearances the only acceptable samples were the two 14% alcohol

preserved samples, however were bitter in aftertaste due to strong alcohol odour and flavour. Thus the following recommendations were made

for trial 3 processing prototypes; only dry salt to be used with 5% concentration and to be stored at -50C and a further reduction of alcohol

concentration to 8% and 11% respectively and both to be stored at ambient temperature.



Further insights into the reactions taking place during shelf life determination continued with the three chemical analyses; water activity, salinity

and pH as shown in Table 6 below. Results showed that slight changes in pH, salinity and water activity had been observed. The alum 5.3% brine

stored at -1.50C and 1.70C revealed a slight reduction in pH and increased salinity over 22 days of storage while both the alum 10% dry salt stored

at -1.50C and 1.70C had slight reduction in pH and increased salinity. Both the 14% alcohol samples also showed a reduction in pH over the 64

day period. Note that water activity was conducted only on day 1 to assess the effectiveness in the level of preservatives used.


Type of

test



D1 D15 D22 D1 D15 D22 D1 D15 D22 D29 D43 D64 D1 D15 D22 D29 D1

5

D22 D29 D43 D64 D1 D15 D22 D29 D43 D64

Water Activity

0.96

5@2

3.8

0.96

8@2

3.3

0.82

1@2

4.3

0.91

6@2

4.3

0.93

5@2

4.0

Salinity 8.3 8.6 8.6 >10.

5

9.2 10.5 >10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.5 >10

.5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

>10.

5

pH 7.16 6.53 6.75 6.99 6.72 6.28 7.71 7.65 6.46 6.26 6.22 6.10 6.7 7.53 6.40 6.13 7.66

6.32 6.27 6.32 6.51 7.51 7.35 6.43 6.19 6.35 6.43

Based on combined tests results; microbial, descriptive profile, hedonic scale and chemical analyses suggestions made to further improve the

quality and shelf of the gonad products were as follows; only dry salt to be used with 5% concentration and to be stored at -50C and a reduction

in alcohol concentration at 8% and 11% respectively and both to be stored at ambient temperature. These suggestions were implemented in

trial 3 processing indicated below.



6.3.1 Trial 3 Processing Design

Trial 3 processing involved the processing of 4 prototypes: preserved in 5% dry salt, dextrin and alginate coating prior to storage at -5.40C; and

8% and 11% alcohol concentrations to be stored at ambient temperature then all to be stored for a total of 34 days as shown in the experimental

flow diagram in Figure 6 below.



The four prototypes were tested for shelf life over a period of 34 days using the following indicators; microbial determinations, organoleptic

assessments and chemical analyses. These tests were carried out on preserved samples that were stored at -5.40C for dry salt and dextrin and

alginate coated samples while the 8% and 11% alcohol samples were both to be stored at ambient temperature of 300C as indicated in Table 9

below.


5% dry salt 8% alcohol Dextrin and Alginate coating 11% alcohol

Stored @ -5.40C

Stored @ ambient temperature



Similar to trial 2, only two microbial analyses; aerobic plate count and psychrophilic bacterial count were conducted in the shelf life prediction

of gonads preserved in 5% dry salt and dextrin-alginate coated stored at -5.40C and the 8% and 11% alcohol stored at ambient temperature for

a period of 34 days. Results as indicated in Table 9 below showed that day 1 microbial levels of gonads were quite high which may be due to

cross contamination from the 3.5% brine used. The 3.5% brine was this time manually fetched from a mobile cool storage parked outside the

building about 30 meters away from the processing room compared to manually fetched from the cool room next to the processing room.

Furthermore, on the processing day, delivery and weighing of Tahitian nuts (ivi) was made at the same entrance where the 3.5% brine was

brought through hence the likelihood of cross-contamination. However, it appears that microbial levels decreased over time to acceptable levels

on all the four prototypes on days 15 and 34.


Type of test -5.40C (-50C) Alcohol @ ambient tempt

5% dry salt Dextrin & alginate 8% 11%

D 1 D 15 D 34 D 1 D 15 D 34 D 1 D 15 D 34 D 1 D 15 D 34

SPC (CFU/g) or EAPC/g-<300)

6.1×103

1.0×103

5.0×102

3.6×102

2.2×102

5.9×105

1.4×104

3.8×103

50

3.0×103

4.2×103

5.1×103


1.4×104

7.0×102

1.1×103

2.0×103

ND

1.0×102

1.0×102

1.7×103

ND

1.0×102

1.5×102

ND

ND = Detection limit is 100 colonies, EPAC – estimated plate aerobic count is referred to <300 counts for Standard Plate Count (SPC) and <2500 counts for

psychrophilic bacteria.

It is important to note that the dextrin-alginate coated sample stored at -5.40C had the highest level of standard plate count on day 34.

Confirmation of this unacceptable level was observed on day 34 organoleptic assessments discussed below. This may indicate the importance

of employing multiple indicators in the shelf life prediction protocol especially organoleptic assessment.



Similar to trials 1 and 2, organoleptic assessments used included descriptive profiling and hedonic scaling for each prototype stored in two

different temperatures respectively as indicated in Tables 11 and 12 below. Table 11 shows that the dextrin-alginate coated samples lost its

acceptable characteristics on day 15; hence the estimated shelf life was 10 days similar to 5.3% brine in trial 1 processing.

Table 11: Organoleptic Evaluation of Gonads from Descriptive Profile of Gonads from Processing Trial 3

Type of test --5.40C Alcohol @ ambient tempt (30.60C)


D 1 D 15 D 34 D 1 D 15 D 34 D 1 D 15 D 34 D 1 D 15 D 34

Colour Bright Bright Dull Bright Fade dark

Dull Bright Bright Bright Bright Fade dark

Dark

Texture Firm Firm Soft and melted

Firm Soft Melted Firm Firm Firm Firm Firm coagulated

Firm

Odour Fresh seaweed

Seaweed Strong sea weed

Mild seaweed

Neutral Weak sea weed

Alcohol Alcohol Weak fermented alcohol

Alcohol Strong alcohol

weak alcohol

Taste & Flavor Seaweed sweet salty

Seaweed sweet & abit salty

Mild sea weed

Sweet & mild seaweed

Neutral

Neutral Sweet & salty

Sweet salty alcohol

Umami taste (acceptable)

Strong alcohol

Strong alcohol

Weak fermented alcohol

Aftertaste seaweed sweet Slightly bitter

Sweet none Bitter alcohol alcohol alcohol Salty & bitter

Strong alcohol

Slight alcohol

Out of the four prototypes, only alcohol preserved samples retained the acceptable until day 34. These were the 8% and 11% alcohol stored at

ambient temperature that retained bright colour, firm texture, and the acceptable alcohol flavour. Given the data obtained to date, both of

these alcohol preserved samples could have a much longer shelf life over 34 days. For the 5% dry salt sample, its acceptable characteristics was

achieved on day 15, however these characteristics were reduced on day 34, hence its shelf life was estimated as 23 days.

These acceptable characteristics were confirmed by the hedonic scaling as indicated in Table 12 below which revealed that out of the four

prototypes, the day 1 dextrin-alginate coated stored at -5.40C was ranked 1st while the 5% dry salt stored at -5.40C and the 8% alcohol both


ranked 2nd. On day 15, the 5% dry salt stored at -5.40C and the 8% alcohol stored at ambient temperature both ranked 1st while 11% alcohol

stored at ambient temperature ranked 3rd. On day 34, the 8% alcohol ranked 1st, while 11% alcohol and the 5% dry salt both ranked 2nd. The

dextrin-alginate coat was rejected on day 34.

Table 12: Hedonic Evaluation of Gonads from Processing Trial 3

Type of test -5.40C Alcohol @ ambient tempt (30.60C)


D 1 D 15 D 34 D 1 D 15 D 34 D 1 D 15 D 34 D 1 D 15 D 34

Colour 3.3 4.5 3.0 4.2 3.5 2.0 4.3 4.0 3.5 3.8 3.5 3.0

Texture 4.3 4.5 3.0 3.3 2.0 2.5 3.5 3.5 4.0 2.8 3.0 3.0

Odor 3.5 4.5 3.0 4.2 2.5 2.5 4.3 4.5 4.5 4.2 4.5 4.5

Taste & Flavor 3.7 4.5 4.0 4.2 2.0 3.5 3.0 5.0 5.0 2.2 3.5 4.0

Aftertaste 4.2 4.5 3.5 3.8 2.0 3.5 3.7 5.0 4.5 2.3 3.0 3.0

Overall ranking 2nd 1st 2nd 1st 4th 4th 2nd 1st 1st 4th 3rd 2nd

Furthermore, based on the collective hedonic scale and organoleptic assessments, the dextrin-alginate sample stored at -5.40C became

unacceptable resulting in its shelf life to be 10 days and the 5% dry salt estimated shelf life for 23 days. Similarly, the two alcohol preserved

samples increased their acceptance characteristics as the alcohol and the gonads mature over time and began to ferment producing umami

flavour on day 34 for the 8% alcohol while the 11% alcohol had yet to reach the umami stage hence have a much longer shelf life beyond the 34

days.

For the purpose of finalizing the result of trial 3, a further processing is recommended. This would involve the processing of 5% dry salt to be

stored at -50C and an 8% alcohol to be stored at ambient temperature. These two recommended formulations are expected to include

consumer acceptance tests as well inter-laboratory analyses for the confirmation of the in-house tests been conducted to date.



Like trials 1 and 2, the three chemical analyses used were water activity, salinity and pH as shown in Table 13 below. Results showed that slight

reduction in pH, salinity could not be measured further due to the maximum limit of detection in the refractometer used. Like trial 2, note that

water activity was conducted only on day 1 to assess the effectiveness in the level of preservatives used.


Type of test -50C (-5.40C) Alcohol @ ambient tempt (290C)


D 1 D 15 D 36 D 1 D 15 D 36 D 1 D 15 D 36 D 1 D 15 D 36

Water Activity [email protected] C

[email protected]

[email protected]

[email protected]

Salinity (%) >10.5 >10.5 >10.5 8.3 >10.5 >10.5 >10.5 >10.5 >10.5 >10.5 >10.5 >10.5

pH 6.51 6.81 6.54 6.46 6.39 5.96 6.51 6.15 6.17 6.35 6.29 6.14

The combined tests results from microbial, descriptive profile, hedonic scale and chemical analyses suggest the use of the following;

5% dry salt to be processed and then stored at -50C and

8% alcohol with 5% dry salt and then stored at ambient temperature

These are to be implemented in the last round and final processing consumer acceptance tests and inter-laboratory analyses are to be conducted

for the confirmation of the in-house tests carried out to date.

7.0 Limitations of the study

Two major limitations were identified to have affected the quality of the processed sea urchin gonad products for this project.

1. Spawning time of sea urchin. Unless the spawning time of sea urchin is clearly identified, harvesting and processing at the right time

would provide acceptable size and colour in the production of high quality gonad products. This I think should be clearly identified and

confirmed including the harvest sites that produces yellow and orange gonads. This was clearly evident in the amount of rejects made

mailto:[email protected]

mailto:[email protected]


during processing trials 1 and 3 and the gonad yields produced. This perhaps suggests further investigations and research in the area to

be conducted.

2. The availability of reliable cool and supercooling storage facilities at -50C. This was clearly evident in the variation of temperature recorded

by the temperature data logger. Even though effort was made in the transfer of samples to other facilities, it was later found that the

same issue existed in such facilities. There had high variations in the temperature of the facilities which may have affected the estimation

of the shelf life of sea urchin gonad products. This may mean that in the proposed final processing, a -50C supercooling facility is to be

made available before the processing takes place which may also then determine the shelf life of the gonad products at such a stable

temperature.

8.0 Conclusion

The project revealed that yellow and orange colored sea urchin gonads could be preserved to achieve the most acceptable and desirable

organoleptic characteristics; bright mango-orange or yellow colour, whole firm texture, fresh seaweed odour, fresh seaweed-sweet taste and

free of leaking fluids using 5% dry salt stored at -5.40C with the shelf life of 23 days and the 8% alcohol mixed with 5% dry salt stored at ambient

temperature with the shelf life of 34 days. These were obtained from trial 3 processing after series of evaluations of trials 1, 2 and 3 processing

procedures and formulations. It is interesting to note that brining appear to be unacceptable due to the oozing and leaking of yellow and orange

fluid of Tripneustes gratilla species into the brine contributing to milky-turbid solution. Soaking in sodium phosphate did not stop the oozing,

instead aggravated milkiness and turbidity in brined samples. Hence the dry salting and alcohol based preservations were the most suitable

preservations identified at this stage. This therefore needs further confirmation by conducting further inter-laboratory analyses and consumer

acceptance tests in the next and final round of processing.

9.0 References

Chen, Y; Chen, T; Chiou, T and Hwang, D. (2013). Seasonal variation on general composition, free amino acids and fatty acids in the gonad of

Taiwan’s sea urchin Tripneustes gratilla. Journal of Marine Science and Technology, 21:6; 723-732.

Fiji Ministry of Health. (2010). Food Safety Regulation 2009. Fiji Islands Government Gazette Supplement. Government Printer, Suva. Pg 246.

Health Protection Agency. (2009). Guidelines for assessing the microbiological safety of ready-to-eat foods. London. Pg 33


James, P.J; Heath, P and Unwin, M.J. (2007). The effects of season, temperature and initial gonad condition on roe enhancement of the sea

urchin Evechinus chloroticus. Aquaculture, 270; 115-131.

Kato, S and Schroeter, S.C. (1985). Biology of Red Sea Urchin, Strongylocentrotus franciscanus, and its fishery in California. Marine Fisheries Review, 47(3); 1-20.

Kato.S,(1972). Sea urchins: a new fishery develops in California. Mar. Fish. Rev, 34(9-10), 23-30.

Lawrence, J. (2013). Sea Urchins: Biology and Ecology (2013). Elsevier, London. PP 270.

Manuel, J.I; Prado, V.V; Tepait, E.V; Estacio, B.M; Galvez, G.N and Rivera, R.N. (2013). Growth performance of the sea urchin, Tripneustes gratilla in

cages under La Union Condition, Philipines. E-International Scientific Research Journal, V (1); 195-202.

McManus, L.T; Gomez, E.D; McManus, J.W and Juinio, A. (Undated). Sea urchin management in Bolinao, Pangasinan, Philipines: Attempts on

sustainable use of communal resource.

Reynolds J.A and Wilen J.E. (2000). The sea urchin fishery: Harvesting, processing and the market. Marine Resource Economics, 15; 115-126.

Schlosser, S.C; Lupatsch, I; Lawrence, J.M; Lawence, A.L and Shpigel, M. (2005). Protein and energy digestibility and gonad development of the

European sea urchin Paracentrotus lividus fed algal and prepared diets during spring and fall. Aquaculture Research, 36; 972-982.

Shpigel, M; Schlosser, S.C; Ben-Amotz, A; Lawrence, A.L and Lawrence, J.M. (2006). Effects of dietary carotenoid on the gut and the gonad of the sea urchin Paracentrotus lividus. Aquaculture, 261; 1269-1280.

Sonu, S.C. (2003). The Japanese sea urchin market: NOAA technical memorandum NMFS. U.S. Department of commerce. Southwest Region. P 34.

Suckling, C.C; Symonds, R.C; Kelly, M.S. and Young, A.J. (2011). The effect of artificial diets on gonad colour and biomass in the edible sea urchin Psammechinus miliaris. Aquaculture 318; 335-342.

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